ISOLATION AND CHARACTERIZATION OF ANTIMICROBIAL COMPOUNDS FROM THE PLANTS, ERYTHRINA ABYSSINICA DC. AND CHASMANTHERA DEPENDENS HOCHST IRUNGU STANLEY [B.Ed, Sc] 156/13105/05 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE IN THE SCHOOL OF PURE AND APPLIED SCIENCES, KENYATTA UNIVERSITY September, 2012 DECLARATION DECLARATION BY CANDIDATE This thesis is my original work and has not been presented for any other degree in any other university. Irungu Stanley Department of Chemistry Signature …………….……………………………Date ……………………… DECLARATION BY SUPERVISORS This thesis has been submitted in partial fulfillment of Masters of Science degree of Kenyatta University with our approval as supervisors. Dr. Alex K. Machocho Department of Chemistry Kenyatta University Signature …………….……………………….Date ……………………………… Prof. Paul K. Tarus Department of Chemistry and Biochemistry Chepkoilel University College (Moi University) Signature ……………….……………………….Date ……………………………… ii DEDICATION To my parents, my wife Rachael and children Sharon and Macfarland iii ACKNOWLEDGEMENT I do acknowledge my supervisors, Prof. P. Tarus of Chepkoilel University College (Moi University) and Dr. A. Machocho of Kenyatta University for their continued advice, encouragement and patience throughout this research and writing of the thesis. Many thanks to entire Department of Chemistry staff at Kenyatta University for the help they accorded me especially Mr. Elias Maina who supported me in technical aspects of laboratory equipments. My appreciation also goes to Dr. Christine Bii of Kenya Medical Research Institute and Mr. Ernest Ruto of Public Health Laboratory for the guidance in bioassay tests and my classmates at Kenyatta University with whom we shared academic challenges and inspired each other. iv TABLE OF CONTENTS PAGE Declaration ii Dedication iii Table of contents v Acknowledgment iv List of plates ix List of tables x List of schemes xi List of abbreviations xii Abstract xiv CHAPTER ONE INTRODUCTION 1.1 Background 1 1.2 Economics of herbal medicine trade 10 1.3 Bacteria and fungal infections 11 1.4 Antibiotics and antifungals 13 1.5 Bacteriostatic and fungiostatic agents 14 1.6 Bactericidal agents 16 1.7 Antibiotic resistance 18 1.8 Justification 20 1.9 Statement of the problem 20 1.10 Hypotheses 20 v 1.11.1 General objective 21 1.11.2 Specific objectives 21 CHAPTER TWO LITERATURE REVIEW 2.1 Genus Erythrina 22 2.2 Genus Chasmanthera 31 CHAPTER THREE METHODOLOGY 3.1 Glass ware 39 3.2 Laboratory equipments and instruments 39 3.3 Nuclear magnetic resonance (NMR) 39 3.4 Chromatographic materials 40 3.5 Chemicals 40 3.6 Spray reagents 40 3.7 Collection and preparation of plant materials 41 3.8 General procedure of extraction 41 3.9 Fractionation of the extracts 42 3.10 Compound IS/EA/E/15 (131) 43 3.10.1 Compound IS/EA/D/5 (132) 44 3.10.2 Compound IS/EA/D/3 (134) 45 3.10.3 Compound IS/CD/D/17 (137) 46 3.11 Microbial test cultures 47 3.12 Anti-bacterial screening tests 48 vi 3.13 Anti-fungal screening tests 48 3.14 Minimum inhibitory concentrations (MIC) and minimum bactericidal/fungal Concentrations (MBC/MFC) 49 3.15 Disc Diffusion and MIC rating of the extracts 49 3.16 Structure elucidation 50 3.19 Physical and spectroscopic data of the isolated compounds 50 CHAPTER FOUR RESULTS AND DISCUSSION 4.1 Crude extract yields 53 4.2 Antibacterial and antifungal assay for the extract 54 4.2.1 Antibacterial activity of E. abyssinica 55 4.2.2 Antifungal activity of E. abyssinica 55 4.2.3 Antibacterial activity of C. dependens 55 4.2.4 Antifungal activity of C. dependens 55 4.3 Antibacterial and antifungal activity of the isolated compounds 56 4.6 Minimum inhibitory concentration (MIC) of active crude extracts and the pure compounds 57 4.7 Disc diffusion and MIC rating of the extracts 58 4.8 Structure elucidations 59 4.8.1 Compound 131 59 4.8.2 Compound 132 61 4.8.3 Compound 134 65 4.8.4 Compound 137 68 vii CHAPTER FIVE CONCLUSION AND RECOMMENDATION 5.1 Conclusions 72 5.2 Recommendations 73 References 74 Appendices 85 viii LIST OF PLATES PAGE Plate 1: Photograph of aerial parts of Erythrina abyssinica 24 Plate 2: Photograph of Chasmathera dependens 32 ix LIST OF TABLES PAGE Table 1: Masses of plant material obtained 53 Table 2: Masses of sequential extraction of E. abyssinica and percentage yields 53 Table 3: Masses of sequential extraction of C. dependens and percentage yields 54 Table 4: The inhibition zones (in mm) of the crude extracts 56 Table 5: The inhibition zones (in mm) of the isolated compounds 57 Table 6: The MIC of active extracts 58 1 Table 7: H NMR data (400 MHz, CD3OD) for compound 131 60 13 Table 8: C NMR data (100 MHz, CD3OD) for compound 131 61 1 Table 9: H NMR data (400MHz, CD3OD) for compound 132 62 13 Table 10: C NMR data (100MHz, CD3OD) for compound 132 64 1 Table 11: H NMR data (400 MHz, CD3OD) for compound 134 65 13 Table 12: C NMR data (100MHz, CD3OD) of compound 134 67 1 Table 13: H NMR data (400MHz, CDCl3) for compound 137 69 13 Table 14: C NMR data (100MHz, CDCl3) for compound 137 71 x LIST OF SCHEMES PAGE Scheme 1: Isolation of compound IS/EA/E/15 (131) from EtOAc roots barks extract of E. abyssinica 44 Scheme 2: Isolation of compound IS/EA/D/5 (132) from DCM stems barks extract of E. abyssinica 45 Scheme 3: Isolation of compound IS/EA/D/3 (134) from DCM stem barks extract of E. abyssinica 46 Scheme 4: Isolation of compound IS/CD/D/17 (137) from DCM extract of the stem of C. dependens 47 xi LIST OF ABBREVIATIONS AND ACRONYMS µg Microgram 13C NMR Carbon-13 Nuclear Magnetic Resonance 1H NMR Proton Nuclear Magnetic Resonance CC Column Chromatography COSY Correlation Spectroscopy d Doublet DCM Dichloromethane DEPT Distortionless Enhancement by Polarization Transfer DMSO Dimethyl Sulfoxide EtOAc Ethylacetate HMBC Heteronuclear multiple bond correlation HSQC Heteronuclear single quantum Coherence J Coupling constant KEMRI Kenya Medical Research Institute m Multiplet MBC Minimum Bacterialcidal Concentration MDRC Multiple Drug Resistant Strain MeOH Methanol MFC Minimum Fungicidal Concentration MHA Mueller Hinton Agar MIC Minimum Inhibitory Concentration NA Nutrient Agar xii NMR Nuclear Magnetic Resonance NOESY Nuclear Overhauser Enhancement Spectroscopy PDA Potatoes Dextrose Agar ppm parts per million PTLC Preparative Thin Layer Chromatography s Singlet SDA Sabouraud Dextrose Agar t Triplet TLC Thin Layer Chromatography UN United Nations UV Ultra Violet VLC Vacuum Liquid Chromatography WHO World Health Organization δ Chemical shift xiii ABSTRACT Infectious diseases account for approximately one-half of all deaths in tropical countries. In industrialized nations, despite the progress made in understanding of microbiology and their pathogenic control, incidences of epidemics due to drug resistant micro-organisms and the emergence of hitherto unknown disease causing microbes, pose enormous public health concerns. Thus, there is need to continue the search for appropriate drugs for anti-microbial treatment without much drawbacks. Historically, plants have provided a good source of anti- infective agents. They are cheap and effective in the treatment of diseases while simultaneously mitigating many of the side effects that are associated with synthetic anti- microbial agents. Phytomedicines derived from plants have shown great promise in the treatment of intractable infectious diseases including opportunistic human immunodeficiency virus (HIV) / acquired immune deficiency (AIDS) infections. The plant species Erythrina abyssinica DC. and Chasmanthera dependens Hoschst have been reported to exhibit remarkable antimicrobial activities and are used as herbal remedies by traditional medical practioners. In this study, the plant extracts from the two species were screened for their antibacterial activity against selected strains of bacteria, including Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus, antifungal activities against Trichophyton mentagrophytes and yeasts (Candida albicans). The isolation and purification of bioactive compounds was done using solvent partition and chromatography: CC, PTLC and VLC. Structure characterization was carried out using standard spectroscopic methods: 1H NMR, 13C NMR, DEPT, COSY, HSQC, HMBC and NOESY. 1,5,4′- trihydroxy-5′-prenylchalcone (131), 2',3′,4′,7-tetrahydroxy-5'-prenylflavanone (132) and 2'- methoxy-nor-glycyrrisoflavanone (134) were isolated from Erythrina abyssinica. 2,3- epoxycolumbin (furano diterpenoid) (137) was isolated from Chasmanthera dependens. One compound 2'-methoxy-nor-glycyrrisoflavanone (134) showed high activity against the gram- positive bacteria Staphylococcus aureus with inhibition zone of 15 mm. The DCM crude extract of E. abyssinica stem barks had activity against Staphylococcus aureus and the fungi Trichophyton mentagrophytes with inhibition zones of 10 and 16 mm, respectively. The hexane extract of root tuber of C. dependens was highly active against Staphylococcus aureus and Trichophyton mentagrophytes with inhibition zones of 16 and 23 mm, respectively. The study demonstrated that there is need for further investigation and isolation of more pure compounds from both plants. In addition the crude extracts and the pure compounds be subjected to further bioassay against other disease causing microbes. xiv xv CHAPTER ONE INTRODUCTION 1.1 Background Infectious diseases are number one cause of death world wide accounting approximately one- half of all deaths in tropical countries. Perhaps it is not surprising to see these statistics in developing nations, but what may be remarkable is that infectious disease mortality rates are actually increasing in developed countries such as the United States of America. Death from infectious disease in U.S.A ranked 5th in 1981 and a 3rd leading cause of death in 1992 with an increase of 58% (Pinner et al., 1996). At the same time man’s population is subject to natural atrocities of famine, wars, and infections. Infectious diseases in man are caused by parasitic animals or plants which are derived directly from human or more rarely animal sources.